Cryptic female choice by female control of oviposition timing in a soldier fly

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Behavioral Ecology Advance Access published June 17, 2009

Behavioral Ecology doi:10.1093/beheco/arp083

Cryptic female choice by female control of oviposition timing in a soldier fly

Flavia Barbosa

Division of Biological Sciences, University of Missouri, 205 Tucker Hall, Columbia, MO 65211, USA

There is substantial evidence that cryptic female choice (CFC) is present in numerous taxa. Several mechanisms have been proposed for CFC; however, we only have experimental evidence for a few of them. Female control of oviposition timing is a potentially widespread mechanism of CFC, but it has never been experimentally demonstrated. The aims of this study are to test 2 critical predictions of the hypothesis that CFC through control of oviposition timing occurs in the soldier fly Mewsargus cingulatus: 1) to determine if M. cingulatus females are less likely to oviposit immediately after mating when the male does not perform copulatory courtship than when he does and 2) to determine if failure to immediately oviposit by the female results in lower reproductive success for the male she just mated with. To answer the first question, I compared the oviposition behavior of females that mated with control males versus females that mated with manipulated males that could not perform copulatory courtship. I showed that M. cingulatus females fail to oviposit immediately after copulation when males do not perform copulatory courtship. To answer the second question, I showed that there is last male sperm precedence in M. cingulatus. Because the last male to mate fertilizes most of the female's eggs, a male will benefit when females oviposit immediately after mating with him and before remating with another male. Key words: copulatory courtship, cryptic female choice, oviposition timing, postcopulatory sexual selection, soldier fly, Stratiomyidae. [Behav Ecol]

Postcopulatory sexual selection is potentially important in polygamous species, where the number of copulations by males does not necessarily predict their reproductive success.

There are 2 primary mechanisms: sperm competition and cryptic female choice (CFC). CFC is a female-controlled, postcopulatory process that biases paternity toward males that have a preferred trait over males that lack that trait, when the female has mated with both (Eberhard 1996). Whereas there is abun- dant evidence for sperm competition (Birkhead and Moller 1998; Simmons 2001), there are far fewer studies that provide direct evidence for CFC (see Table 1). However, there is substantial indirect evidence for CFC in numerous taxa (Eberhard 1996).

Several potential mechanisms have been proposed for CFC (Eberhard 1996). Although indirect evidence suggests that there may be more than 20 mechanisms, there is experimental evidence for only a few: female adjustment of the number of eggs laid, female control of copulation duration and sperm transfer, and internal sperm manipulation by the female (see Table 1). Other potential mechanisms have not been demonstrated but are likely to be widespread. One such mechanism is female control of oviposition timing. A female can bias paternity toward a male by ovipositing soon after mating with him and before she mates with another male, if last male sperm precedence occurs (i.e., the last male to mate with a female fertilizes most of her eggs). This means that if a female mates with a second male before ovipositing, she will greatly decrease the first male's reproductive success. Female control of oviposition timing is a potentially important mechanism of CFC because last male sperm precedence is widespread among insects (Thornhill and Alcock 1983). How- ever, there are no known examples of CFC through oviposition timing. The goal of this study is to experimentally test Address correspondence to F. Barbosa. E-mail: fabdm2@mizzou.

edu.

Received 17 February 2009; revised 22 May 2009; accepted 23 May 2009.

For permissions, please e-mail: [email protected]

the hypothesis that CFC by female control of oviposition timing occurs in the soldier fly Merosargus cingulatus (Diptera:

Stratiomyidae).

In M. cingulatus, copulations occur at oviposition sites such as piles of rotting fruit or recently cut grass, in which the larvae develop, and where males patrol and defend a territory. Ovi- position sites attract large numbers of males and females. Male territories are relatively small, with neighboring males typically about 10-15 cm apart (Barbosa F, personal observation). The male attempts to grab and copulate with any female that flies near his territory. Females do not appear to have any oppor- tunity to choose a mate and do not seem to resist mating once they are grasped by a male. It is unclear if females come to the territories primarily for mating or oviposition. Males perform copulatory courtship throughout the copulation. Copulatory courtship involves 2 distinct behaviors: Males tap the female's abdomen with their hind legs and wave their legs in the air.

After mating, females usually lay eggs deep in the rotting veg- etable matter in or close to the male's territory. When females leave a male's territory without ovipositing, they will likely mate again before they have the chance to oviposit elsewhere. Male density at the oviposition sites is high, and if a female is detected by a male, he will likely grab her and mate (Barbosa F, personal observation). Observations of marked animals show that both males and females mate multiply in the field (Barbosa F, personal observation).

The aims of this study are to test 2 critical predictions of the hypothesis that CFC through control of oviposition timing occurs in M. cingulatus: 1) to determine if females are less likely to oviposit immediately after mating when the male does not perform copulatory courtship than when he does and 2) to determine if failure to immediately oviposit by the female results in lower reproductive success for the male she just mated with. To answer the first question, I conducted a field experi- ment where I compared the oviposition behavior of females that mated with control males versus females that mated with manipulated males that could not perform copulatory courtship. I showed that M. cingulatus females fail to oviposit immediately after copulation when males do not perform copulatory

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Behavioral Ecology

Table 1

Studies of CFC where the postcopulatory mechanism could be attributed to the female and was shown to affect male reproductive success

Organism Female mechanism Male trait favored by female References

Cucumber beetle, Diabrotica undecimpunctata howardi

Controlling male access to bursa copulatrix and spermatophore transfer

Water strider, Gerris lateralis Clutch size adjustment Scorpion fly, Harpobittacus nigriceps Clutch size adjustment Guppy, Poecilia reticulate

Water frog, Rana lessonae-esculenta Yellow dung fly, Scathophaga stercoraria

Spider, Arglope keyserlingi Flour beetle, Tribolium castaneum Fly, Dryomyza anilis

Field cricket, Gryllus bimaculatus Decorated cricket, Gryllndes sigillatus

Controlling amount of retained sperm

Clutch size adjustment Unknown

Controlling copulation duration by mate cannibalism

Controlling sperm transfer and storage

Transferring sperm to singlet spermatheca, which is primarily used when ovipositing Clutch size adjustment Controlling duration of spermatophore attachment and therefore of sperm transfer

Intensity of copulatory courtship Tallamy et al. (2002)

Larger body size Arnqvist and Danielsson (1999) Large body size and large nuptial Thornhill (1983)

gift prey item (which are correlated)

Relatively colorful males Pilastro et al. (2004) Not hybrid males Reyer et al. (1999) PGM genotype (preference varies Ward (1998, 2000, 2007) with environment)

Small body size Elgar et al. (2000)

Intensity of copulatory courtship Edvardsson and Arnqvist (2000) and Bloch-Qazi (2003) Number of postcopulatory genital Otronen and Siva-jo thy (1991) taps

Higher dominance status Large spermatophylax size

Bretman et al. (2006)

Sakaluk and Eggert (1996) and Sakaluk (1997)

courtship. To answer the second question, I tested for last male sperm precedence using amplified fragment length polymorphism (AFLP) markers. I showed that the last male to mate with a female fertilizes most of her eggs. This means that a male will benefit when females oviposit immediately after mating with him and before remating with another male.

METHODS

Copulatory courtship

All field observations and experiments were conducted at the Smithsonian Tropical Research Institute field station in Gamboa, Panama, in August 2006 and in July 2007. I set up piles of decomposing fruit peels in open areas by small patches of forest in a residential area. Both male and female M. cingulatus were attracted to the fruit. I captured a total of 62 males. Half of them were treated so that they could not perform copulatory courtship by applying black acrylic paint to their hind legs. This treatment made the legs stiff and prevented normal movement during copulatory courtship;

I did not observe any other change in behavior caused by this treatment, and they obtained mates as readily as untreated males. The other half of the males received the control treatment: They were caught and handled like the manipulated males, but their hind legs were not painted, allowing for normal movement during copulatory courtship. All males were uniquely marked on the thorax with acrylic paint to allow individual identification and were released immediately after being processed. When males returned to their territories, I observed them until they mated. Not all the marked males returned to their territories and mated; I only collected data from the ones that did so. I filmed the copulation and the female behavior until she left the opposition site and then collected the male. I allowed each male to mate only once.

Sperm precedence

I collected mated pairs in the field and raised their offspring for paternity analysis in order to assess last male sperm pre-

cedence. Because all individuals were field caught, their previous mating history is unknown. I observed copulations and afterward watched the female as she oviposited. I then collected the male and female and the piece of fruit on which she had oviposited. Because the fruit peels came from fresh fruit and I allowed them to decompose in sealed enclosures, the only eggs present were those laid by the female that I observed mating. I allowed the eggs to develop to 3-week-old larvae and preserved the adults and these larvae in 100%

ethanol. I collected a total of 9 "families" (the mated pair and the larvae resulting from that clutch), which had an average of 22.77 offspring per family. Twenty offspring per family were randomly picked for the paternity analysis for the families that had more than 20 offspring; all offspring were analyzed in 4 families with fewer than 20 offspring (number of larvae = 7, 8, 19, and 19).

DNA extraction

I extracted DNA from the tissue from flight muscles and legs of adults. For the larvae, I removed the head and used the rest of the body. I froze the tissues with liquid nitrogen and macerated them with a lysis buffer (250 mM Tris—pH 7.5, 2 M NaCl, 100 mM ethylenediaminetetraacetic acid (EDTA), 2.5% sodium dodecyl sulfate and 2% proteinase K), incubating for 2 h at 58 °C. The DNA was purified with a 10% cetrimonium bro- mide solution, a 24:1 chloroform:isoamyl alcohol solution, and 5 M ammonium acetate. Samples were then treated with a 100 ug/ml solution of RNase A for 30 min at 37 °C. DNA was precipitated with ethanol and resuspended in low TE (0.5 M EDTA, 1 M Tris-HCl). DNA samples were quantified with a NanoDrop N-1000 spectrophotometer and diluted to a final concentration of 50 ng/pl.

Amplified fragment length polymorphism

Generating AFLP markers involved 4 steps. First, I digested the DNA with 2 restriction endonucleases, £coRI and Msel. The fragments generated were then ligated to adaptors, which are

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Barbosa • Female control of opposition timing in a soldier fly

small DNA fragments with sticky ends complementary to the enzyme's cutting sites. These 2 steps were combined in a single reaction. The fragments were then submitted to 2 polymerase chain reactions (PCRs): the preselective and the selective am- plifications, where the adaptor sequence is used to build the primers. The purpose of the PCRs is to amplify only a subset of the fragments by adding 1-3 arbitrary bases to the primer sequence. The primers used in the preselective PCR have 1 bp added, and the selective PCR primer can have up to 3 bases added to its sequence.

The protocol used was adapted from Vos et al. 1995. First, 50 ng of DNA was used in a restriction-ligation reaction (IX T4 DNA ligase buffer with ATP, 0.05 M NaCl, 5 ng/ml bovine serum albumin, 5 U £coRI, 1 U Msel, 1 U T4 ligase, 50 uM Msel adaptor, 5 uM -EcoRI adaptor) and incubated at 37 °C for 2 h. The adaptors were synthesized by Integrated DNA Tech- nologies (Coralville, IA). The sequence of the adaptor was for EcoRI forward: 5-CTC GTA GAG TGC GTA CC-3' and reverse: 5-AAT TGG TAC GCA GTC TAC-3' and for Msel forward: 5-GAC GAT GAG TCC TGA-3' and reverse: 5'- TAC TCA GGA CTC AT-3'. The second step was a preselective amplification. The product from the reaction above was diluted 1:10 with low TE; 2.5 ul of it was combined with 1.5 U of Taq DNA polymerase, 20 ul of 10X Taq DNA polymerase buffer, 1 ul of the preselective amplification primer mix, and 0.5 ul of 10 mM dNTP solution; and this solution was brought to 20 ul with water. The PCR began with a cycle of 72 °C for 2 min; followed by 30 cycles of 94 °C for 30 s, 56 °C for 30 s, and 72 °C for 2 min; and a final cycle of 60 °C for 10 min. The structure of the preselective primers used were 5'- GAC TGC GTA CCA ATT CT-3' for the EcoRI primer and 5'- GAT GAG TCC TGA GTA AC-3' for the Msel primer, and they were both synthesized by Integrated DNA Technologies. The amplification product was diluted 1:5 with low TE and used in a selective PCR. The selective PCR used 1 ul of 10X Taq DNA polymerase buffer, 0.75 U of Taq DNA polymerase, 2 pmol of the Msel selective primer, 0.4 pmol of the EcoKl- selective primer, 1.5 ul of the diluted reaction, and 0.25 ul of 10 mM dNTP solution. This mixture was brought to a vol- ume of 10 ul with water. One primer pair combination was identified from a set of 8 primer pairs as the most polymor- phic and therefore most informative. The Msel primer had 3 selective nucleotides added. It was synthesized by Integrated DNA Technologies, and its structure is 5-GAT GAG TCC TGA GTA ACAT-3'. The EcoRI primer had 2 selective nucleotides added and was fluorescently labeled with 6-flourescein amidite dye. Its sequence is 5-GAC TGC GTA CCA ATT CTG- 3', and it was synthesized by Applied Biosystems (Foster City, CA). Fragments were separated by gel electrophoresis and detected by an ABI 3730 DNA Analyzer, which uses an automated fluorescence-based detection system. Fragment size was accu- rately detected by the addition of an internal lane size standard that was also fluorescent marked (Genescan 600 LIZ).

AFLP analysis

Profiles were visualized and scored for presence/absence of fragments ranging from 60 to 400 bp using Softgenetics Gene- Marker software. For the paternity analysis, I used Famoz software (Gerber et al. 2003) to calculate the average exclusion probabilities and to determine paternity of each offspring through categorical allocation. The logarithm of odds score (logarithm of the likelihood ratio) of each father-offspring pair was calculated and used to determine if the males analyzed were the fathers of the offspring of the females they mated with. The exclusion probability equations for dominant markers can be found at Gerber et al. 2000.

1 0.9 08 0.7 H 06

E 0.5

0.4 0.3 H 0.2

0.1

123456789 Family

Figure 1

P2> or percentage of offspring assigned to the last male to mate with each female. The dotted line shows the P% value of 0.5.

RESULTS

Copulatory courtship

None of the 10 manipulated males performed copulatory courtship. Of the 10 females that mated with manipulated males, none oviposited after mating; all flew away soon after copulation ended. All the 13 control males performed copulatory courtship, and all 13 females that mated with controls oviposited (difference in oviposition behavior of females with control vs. manipulated males: Fisher's Exact test, P < .005).

There was no significant difference in the duration of copulation between manipulated and control males (control group: 95.93 ± 43.97 s [mean ± standard deviation], manipulated group: 121.10 ± 59.49; two-tailed (-test, P = 0.245).

Samples were checked for normal distribution (Shapiro-Wilk,

^control ~ U.OOO, rmanipulated ~ U./40).

Sperm precedence

The AFLP-selective primer pair used generated 127 polymor- phic loci, with an average exclusion probability of 0.996. In the 9 families analyzed, average P2> or percentage of offspring sired by the last male, was 0.839 (range = 0.55-1.00) (Figure 1). Because the females used in the analysis were field caught and their mating history is unknown, it is possible that in the 3 cases where P2 was 1, those females only mated once. The average P2 when these females are excluded from the analysis is 0.759 (1-sample (-test, null hypothesis: P2 = 0.5, t= 4.317, P = 0.008). In either case, these results show that there is last male sperm precedence in M. cingulatus.

DISCUSSION

CFC is a female-controlled process that biases paternity toward males that have a preferred trait, after having mated with males with and without the trait. In M. cingulatus, male copulatory courtship influences female oviposition behavior: Females oviposited immediately after copulating with males that performed copulatory courtship but not with males that performed no copulatory courtship. There is last male sperm precedence in this species, so failure of the female to oviposit before remating will decrease the first male's reproductive success. Because oviposition is under female control, the behavior of female M. cingulatus is an expression of CFC. To my knowledge, this study is the first to demonstrate CFC through female control of oviposition timing.

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Behavioral Ecology

It is important to note that there is an alternative explanation for the observed results that cannot be ruled out with the present data. It is possible that females fail to oviposit in the absence of courtship because sperm transfer (and therefore fertilization) does not happen in that case. Copulatory courtship has been demonstrated to be critical for males to achieve complete penetration (and spermatophore transfer) in a chrys- omelid beetle (Tallamy et al. 2002). To distinguish between the 2 scenarios, it would be necessary to determine whether manipulated males that do not perform courtship transfer sperm to the female. However, it should also be noted that females can oviposit even if the last male they mated with does not transfer sperm because they are likely to have sperm stored from previous matings. Therefore, it seems unlikely that females fail to oviposit due to lack of sperm transfer by manipulated males. Another possibility is that manipulated males transfer less sperm than the control males. In that case, the observed female behavior is still an example of CFC by female control of oviposition timing.

Last male sperm precedence is a widespread pattern among insects (Thornhill and Alcock 1983), which suggests that control of oviposition timing is likely to be a common mechanism of CFC. The possibility that females in other species can in- fluence paternity of their offspring by changing oviposition timing has been overlooked and deserves to be explored.

Copulatory courtship is a male trait that is likely under selection by CFC, such that males performing more or superior courtship benefit from increased paternity. Because copulatory courtship occurs only after copulation has begun, it does not play a role in attracting the courted female. In most species, it also does not seem to function in obtaining a second mating with the female being courted, as males leave after a single copulation (Eberhard 1991, 1994). Copulatory courtship is therefore likely to be a trait that influences paternity after mating.

The widespread occurrence of copulatory courtship in arthro- pods (Eberhard 1991, 1994) suggests that CFC is also widespread. A relationship between copulatory courtship and male reproductive success has been found in 2 other species (see Table 1). However, this is the first study to show CFC of copulatory courtship through an external and easily observable female behavior. This discovery increases the possibilities for future studies because such female behaviors can be easily scored.

This is the first study of CFC conducted in the field, rather than in the laboratory. Using field individuals entails some lim- itations, the main one being that the mating history of those individuals is unknown. However, field experiments provide results that are relevant to natural populations. Female choice can play out very differently in the field and in the laboratory:

Factors such as predation risk and the costs of sampling males can make female preferences more costly, which may result in preferences not being expressed as strongly, if at all. In a laboratory setting, these factors are often removed, which in some species is known to lead to a level of female choosiness that would not be expressed in the field (Jennions and Petrie 1997). Thus, field studies are fundamental to assess the rele- vance of CFC: Only by showing that these female preferences are expressed in natural populations, can we confirm that CFC is an important agent of sexual selection.

FUNDING

Smithsonian Tropical Research Institute; Dorothea Bennett Memorial Graduate Research Fund; Animal Behavior Society.

William Eberhard shared invaluable ideas for the execution of this work. Ulrich Mueller, Robert Snyder, and Lori Eggert provided infor-

mation on the AFLP technique and analysis. Michael Reichert, Rex Cocroft, William Eberhard, Carl Gerhardt, and 2 anonymous reviewers made helpful comments on drafts of the manuscript. I would also like to thank the staff at Smithsonian Tropical Research Institute for their assistance and the Autoridad National del Ambiente at the Republic of Panama for issuing research permits.

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